RNAi and Gene Silencing

Pete Burrows

MIC 759

September 16, 2008

Lecture Outline

• Background/discovery

• siRNA/shRNA

• Movie

• miRNA– Biogenesis– Functions– Applications

# Publications

Focus on RNA interference - A user’s guideSeptember 2006

In situ hybridization for mex-3 mRNA

No probe

anti-sense ssRNA

No RNA

dsRNA

1998 Feb 19;391(6669):806-11

Nature 391:744 1998

Puzzles of the RNAi

• Both sense and anti-sense ssRNA effective

• Catalytic – very few copies of dsRNA could silence abundant mRNA

• Therefore not conventional antisense

• Only dsRNA targeting mature mRNA are effective, not to introns or promoters

• RNAi can cross cellular boundaries

Fire, A, 2006 Nobel Prize Acceptance Speech

Class Length (nt) Function

Micro RNA (miRNA) 19-25 Translational repression

Small interfering RNA (siRNA) 19-21 Target mRNA cleavage

Piwi-interacting RNA (piRNA) 26-31 Transposon control in germ cells

Classes of small RNAs

Class Length (nt) Function Organism

Trans-acting siRNA (tasiRNA)

21-22 mRNA cleavage Plants

Small-scan RNA (scnRNA) ~28 Histone methylation and DNA elimination

Tetrahymena

Repeat-associated siRNA (rasiRNA)

24-27 Transposon control/transcriptional silencing

Yeast, plants, flies

dsRNA

RNAiDicing and slicing

• All RNA silencing pathways are triggered by 21-27 nt long small RNAs– Small interfering RNAs – siRNA– Micro RNAs – miRNA– Piwi RNA

• RNAi induction using long dsRNA only operates in plants and invertebrates

• In mammals, long dsRNA (>30 bp) induces on the IFN response including PKR, inhibits translation, and activation of RNaseL, degrades mRNA

Fire, A, 2006 Nobel Prize Acceptance Speech

PKR Inhibition of Translation

PKR Inhibition of Translation

siRNA and shRNA

• siRNA (short interfering RNA)– typically synthesized chemically then

introduced into target cells

• shRNA (short hairpin RNA)– typically introduced as a plasmid or viral

vector– endogenous production, can be long term– enters the RNAi pathway upstream of siRNA

Novina and Sharp Nature 430:161 2004

Dicer

• Dicer generates RNAs with 2 nt 3’ overhang and 5’ phosphorylated terminus, both required for activity

RISC

• RISC has helicase, endonucelase “slicer”,S and homology searching domains.

• Initial RISC is inactive until transformed into active form by unwinding of the siRNA duplex and loss of sense strand.

Published by AAAS

J. Liu et al., Science 305, 1437 -1441 (2004)

Fig. 1. Only mammalian Ago2 can form cleavage-competent RISC

Identification of Ago2as “Slicer” in the RISC

Published by AAAS

J. Liu et al., Science 305, 1437 -1441 (2004)

Fig. 2. Argonaute2 is essential for mouse development

Published by AAAS

J. Liu et al., Science 305, 1437 -1441 (2004)

Fig. 3. Argonaute2 is essential for RNAi in MEFs

The ago1 mutant Arabidopsis develops abnormally because it does not produce aneffector of silencing. The Argonaute genes were so named because the mutant plantslook like an argonaute squid.

The Sainsbury LaboratoryJohn Innes CentreColney LaneNorwich, NR4 7UH, UK

Summary of siRNAand shRNA processing

Processing of siRNA

• Which becomes guide strand in the RISC and which is excluded?– Sequence and structure– Strand with the less-tightly base pared 5’ end

is incorporated becomes guide strand

miRNA

• Abundant ssRNA from a few thousand to 40,000 molecules /cell

• Found in all metazoans• 0.5-1% of genes• siRNA targets genes from which it is derived in a

sequence specific manner• miRNA regulates separate genes and has

imperfect complementarity. May be 100’s/miRNA. Usually have many binding sites in each 3’ UTR, and several different miRNA can target same 3’ region. Combinatorial control

• 30 – 50 % of genes regulated by miRNA

miRNA

• Many miRNA are embedded in introns of protein encoding genes and are transcribed together with host genes.

• miRNA can be expressed in developmentally tissue specific fashion but may not be expressed in tissues where putative target sequences are.

Plasterk RHA Cell 124:877 2006

Tissue-specificexpression of miRNA

Du, T. et al. Development 2005;132:4645-4652

The structure of human pri-miRNAs

Overview of miRNA biogenesis

Cullen Nature Immunology 7:563 2006

Processing of miRNA

• Long primary Pol II transcript (pri-miRNA)• Cleaved by Drosha, nuclear RNase III endonuclease to

establish one end of the miRNA (pre-miRNA)– Also need dsRNA binding protein Pasha (flies) DGCR8

(humans)

• The pre-miRNA exported from the nucleus by Exportin 5• Cut by Dicer→ miRNA• Strand with the less-tightly base pared 5’ end becomes

mature miRNA, other strand becomes miRNA* and degraded

• Worms and mammals only one Dicer and it makes miRNA and siRNA. Flies have one for each.

Imperfect homology between miRNA and 3’ UTR of target mRNASeed sequence has perfect homology

Players in miRNA biogenesis

• Drosha– Nuclear RNase III enzyme. Initiates miRAN

biogenesis by cleaving pri-miRNA into pre-miRNA

• Pasha– Partner of drosha is a dsRNA binding protein.

Human DGCR8

• Exportin-5– Nuclear transmembrane protein that transports

pre-miRNA form nucleus to cytoplasm. Works in conjunction with GTP-Ran

Players in miRNA and siRNA

• Argonaute (AGO)– PAZ domain binds the characteristic two-base 3'

overhangs of siRNAs – PIWI domain: dsRNA guided hydrolysis of ssRNA– Slicer in RISC

• Dicer (DCR)– Multi domain RNase III enzyme the cleaves dsRNA or

stem-loop pre-miRNA into siRNA and miRNA

• TRBP– Cofactor for Dicer

• RISC– RNA induced silencing complex

Mechanism of miRNA suppression of gene expression

• Transcription

• mRNA degradation

• Translational repression– 1 Initiation– 2 Post-initiation step

• Co-translational degradation of the nascent peptide

Mechanism of miRNA suppression of gene expression

• Translational repression– 1 Initiation– 2 Post-initiation step

How to distinguish?

miRNA can repress and activate translation

miRNA

• miRNA in disease– Loss of function mutation of miRNA– Gain of function mutation of miRNA, e.g

overexpression– Mutation of target site, no longer binds miRNA– Mutation of target site, now binds miRNA– Tumor suppressors– Oncogenes “oncomirs”

In vivo applications of RNAi

• Highly specific– Silence a single nucleotide difference in a dominant

negative allele

• Resistance not (less) a problem– Can design new RNAi if a mutation arises and original

targeted sequence is changed

• Problems– Stability– Delivery– Toxicity

Couzin Science 312:1121 2006 Grimm, et al. Nature 441:537-541 2006

Liver damage in mice expressing shRNA long-term

Off Target Effects

• Global, due to induction of innate immune responses

• Cross reactive, due to sequence homology with other mRNA sequences

• Not easy to recognize unless global gene expression studies performed.

• Good to have multiple target sequences

Toll-like receptors (TLR) canrecognize dsRNA

http://www.glocalbeer.dkhttp://tolweb.org/tree?group=Ascomycota&contgroup=Fungi

Taphrina S. pombe S. cerevisiae Morel Penicillium

Swahili word for beer (Pombe)

Schizosaccharomyces pombe has DCR and AGO but not in Saccharomyces cerevisiae